Compositions and methods for treating cancer

ABSTRACT

The invention provides compositions and methods to treat a hyperproliferative disorder with a GSH synthesis inhibitor and an anti-cancer composition.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under CA172218 awardedby the National Institutes of Health and 2012-DN-130-NF0001 awarded bythe Department of Homeland Security. The government has certain rightsin the invention.

BACKGROUND

Melanoma is a cancer of the skin and is the fastest growing cancerincidence in the world today. Disease detected early can be removed bysurgery, but when melanoma spreads to other parts of the body (calledmetastatic melanoma) it is almost uniformly fatal. The reason for thisis that metastatic melanoma rapidly becomes resistance to all forms oftreatment. The first new therapy that appeared effective for melanomawas approved in 2011. The pharmaceutical called vemurafenib targetspatients with a gene mutation (BRAF^(V600E)) that is present in abouthalf of melanoma patients. Although these patients respond well to thetreatment, melanoma develops resistance to the therapy rapidly. Thus,the new therapy, which initially was heralded as the end of melanoma,extends life expectancy by only months with severe side effects.Vemurafenib is one of several BRAF inhibitors that are being used formelanoma therapy that target the BRAF protein. Melanoma developsresistance to all of these therapies. Several other drugs that havedifferent mechanisms of action are also approved for melanoma treatment,but the disease eventually develops resistance to all therapies formelanoma. There is no treatment for metastatic melanoma that overcomesresistance of melanoma cancer cells, which leads to a high mortalityrate.

Small molecule MAPK inhibitors (MAPKi, e.g. BRAFI: vemurafenib,dabrafenib and MEKi: cobimetinib, tramatinib) have been shown tosignificantly reduce tumor burden, and often delay disease progression.However, resistance to MAPKi is observed (almost invariably) within 6-8months, which limits the overall clinical outcomes for patientsundergoing MAPKi therapies. The precise cellular mechanism that drivesMAPKi resistance in metastatic melanoma is not known. However, evidencesuggests several plausible mechanisms, including re-activation of theMAPK pathway; metabolic re-programming; and stress-induced adaptiveresponses (e.g., autophagy and unfolded protein response).Unfortunately, therapeutic strategies targeting these mechanisms tocircumvent the development of MAPKi resistance have yet to yieldsignificant improvement in clinical outcomes for metastatic melanomapatients.

There is therefore a need for compositions and methods for the treatmentof melanoma that prevents melanoma tumors from acquiring resistance toMAPKi.

SUMMARY

In certain embodiments, the present invention provides the use of aglutathione (GSH) synthesis inhibitor in conjunction with one or moreanti-cancer compositions for the therapeutic treatment of ahyperproliferative disorder. The use of the GSH synthesis inhibitor iseffective in preventing drug resistance in the treatment of metastaticmelanoma or other types of cancer cells.

In certain embodiments, the hyperproliferative disorder is cancer. Incertain embodiments, the cancer is drug-resistant. As used herein, theterm “drug-resistant” is reduction in effectiveness of a drug in killingmalignant cells; reducing cancerous tumor size and rate of growth; andameliorating the disease or condition. In certain embodiments, thedrug's effectiveness is reduced by at least about 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90%, or even 100%, as compared to its effects whenfirst administered to the mammal.

In certain embodiments, the cancer is melanoma. In certain embodiments,the melanoma is resistant to MAPK pathway inhibitors. In certainembodiments, the melanoma is resistant to vemurafenib treatment.

In certain embodiments, a phenyl butyric acid (PBA) or apharmaceutically acceptable salt thereof is administered simultaneouslywith the anti-cancer composition.

In certain embodiments, the GSH synthesis inhibitor and the anti-cancercomposition are administered sequentially.

In certain embodiments, the administration of the GSH synthesisinhibitor begins about 1 to about 10 days before administration of theanti-cancer composition.

In certain embodiments, the administration of the GSH synthesisinhibitor and administration of the anti-cancer composition begins onthe same day and/or simultaneously.

In certain embodiments, the GSH synthesis inhibitor is buthioninesulfoximine (BSO).

In certain embodiments, the anti-cancer composition comprisesvemurafenib.

In certain embodiments, the anti-cancer composition compriseschloroquine (or hydroxychloroquine). In certain embodiments, theanti-cancer composition comprises a derivative of triphenylphosphonium(TPP), or a pharmaceutically acceptable salt thereof.

In certain embodiments, the anti-cancer composition comprises isipilimumab.

In certain embodiments, the PBA or a pharmaceutically acceptable saltthereof is administered in combination with vemurafenib, and the canceris melanoma.

In certain embodiments, the PBA or a pharmaceutically acceptable saltthereof is administered in combination with vemurafenib and chloroquine(or hydroxychloroquine), and the cancer is melanoma.

In certain embodiments, the present invention provides a use of thecombination of a GSH synthesis inhibitor and anti-cancer composition inthe preparation of a medicament for the treatment of ahyperproliferative disorder in a mammal.

In certain embodiments, the present invention provides a kit comprisinga GSH synthesis inhibitor, a container, and a package insert or labelindicating the administration of the GSH synthesis inhibitor with ananti-cancer composition for treating a hyperproliferative disorder.

In certain embodiments, the present invention provides a productcomprising a GSH synthesis inhibitor and an anti-cancer composition; asa combined preparation for separate, simultaneous or sequential use inthe treatment of a hyperproliferative disorder.

In certain embodiments, the present invention provides a method fortreating a hyperproliferative disorder in a mammal, comprisingadministering to the mammal a combination of a GSH synthesis inhibitor;and an anti-cancer composition.

In certain embodiments, the GSH synthesis inhibitor is administered formore than a month.

In certain embodiments, the GSH synthesis inhibitor is administered formore than a year.

In certain embodiments, the GSH synthesis inhibitor or apharmaceutically acceptable salt thereof is administered at a dosage ofat least 0.1/mg/kg/day.

In certain embodiments, the GSH synthesis inhibitor is administered at adosage of at least 100 mg/kg/day.

In certain embodiments, the present invention provides a use of a GSHsynthesis inhibitor and an anti-cancer composition for the therapeutictreatment of a hyperproliferative disorder. In certain embodiments, thehyperproliferative disorder is cancer. In certain embodiments, thecancer is melanoma.

BRIEF DESCRIPTION OF DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1(a)-1(b) are graphs illustrating no significant difference intumor growth rate for untreated controls (FIG. 1(a)) and mice treatedwith BSO alone (FIG. 1(b)).

FIG. 2 illustrates that tumor xenografts in mice acquire resistance forBRAFi treatment alone within 140 days.

FIG. 3 illustrates that the combination of BSO with PLX4720 producesnearly 90% complete and durable tumor responses.

FIG. 4 illustrates that the complete response rate and overall survival(OS) for mice treated with the combination of BSO with PLX4720 wassignificantly higher than mice treated with BRAFi alone.

FIG. 5 illustrates the pharmacokinetics B-14 labeled PBA in mice.Biodistribution of C14-PBA in 451LuBR tumor (N=2).

DETAILED DESCRIPTION

Melanoma is the dangerous type of skin cancer that develops in cellsthat produce melanin (melanocytes), usually presenting as an irregularspot/mole on the skin. Causes of melanoma include UV radiation and agenetic predisposition to this type of cancer. Unlike other cancers,prevalence of melanoma is increasing, with the highest occurrence amongindividuals 25-29 years old. The overall lifetime risk of developingmelanoma is 2.4%. In 2015, 73,870 new invasive melanomas are expected tobe diagnosed, with 9,940 people expected to die of melanoma. With earlytreatment, survival rate is 97%.

Melanoma can migrate to other parts of the body (metastatic melanoma),and one-year survival rate drastically decreases with metastasis—15-20%for Stage IV. Current types of treatment include surgery, immunotherapy(Immune checkpoint inhibitors for advanced melanoma), chemotherapy,radiation therapy, targeted therapy (target cells with gene changes) andBRAF Inhibitors. BRAF is a protein kinase of the mitogen-activatedprotein kinase (MAPK) pathway, and it regulates cell growth,proliferation, and differentiation. Research suggests a BRAF^(V600E)mutation causes the BRAF protein (produced through the MAPK pathway) tobecome oncogenic. The mutation may lead to increased and uncontrolledcell proliferation, and resistance to apoptosis. The BRAF mutation isobserved in about 50% of melanoma tumors. Its presence is associatedwith poor prognosis in metastatic melanoma.

Melanoma is the fastest growing cancer incidence in the United States.Surgery is curative for melanoma confined to the skin, but metastaticmelanoma is lethal. Current FDA approved therapies for metastaticmelanoma (e.g., Vemurafenib, Ipilimumab), have increased life expectancyby months, but resistance develops rapidly. The exact mechanism by whichdrug resistance develops is unclear; however, autophagy is known to playa major role. Autophagy is a self-degradative response of the celltowards nutrient stress. Conversely, autophagy also plays a housekeepingrole by removing mis-folded or aggregated proteins and clearing damagedorganelles by forming autophagosomes. Thus, autophagy is believed toplay an important role in tumor progression and developing drugresistance during later stages of cancer. The Unfolded Protein Response(UPR) in the ER associated protein degradation is one of the pathwaysthat initiates autophagy in stressed cells. UPR involves the activationof three signaling pathways mediated by IRE-1, PERK and ATF6. Thesepathways work towards decreasing the protein load of ER by increasingthe expression of molecular chaperons, activation of ERAD (ER associatedprotein degradation) and autophagy. However if the damage caused by thestress is extensive UPR signaling pathways initiate apoptosis. Amy S.Lee, Cancer Res (2007); 77:3496-3499. Emerging evidence shows that inmalignant cells ER stress can be pro-survival and contribute to thedevelopment of drug resistance by initiating autophagy.

Oncogenic BRAF is a known regulator of cellular metabolism thatactivates adaptive reprogramming through transcription factormicrophthalmia-associated transcription factor (MITF) and co-factorPGC1α in melanoma cells. BRAF inhibition (BRAFi) is also known topromote mitochondrial respiration and increased cellular oxidativestress. Within this context, it is also known that BRAFi-resistantmelanoma cells possess an adapted dependence on mitochondrialrespiration (OXPHOS) and, consequently, an increased sensitivity toinhibitors of mitochondrial respiration. Further, this MAPKi shift incellular metabolism towards OXPHOS and resulting increase in oxidativestate has been implicated with an adapted-cellular program that activelymaintains MAPKi-resistance. The relationship between cellular oxidativestate and thiol (R(G)SH) redox equilibrium (ratio of reduced thiols tooxidized thiols) is well established. In the context of melanoma, thepotential pivotal role in adaptation to BRAFi-induced oxidative stressis supported by emerging evidence that implicates phospholipidglutathione peroxidase 4 (GPX4) activity with melanoma.

The present inventors hypothesized that MAPKi driven changes in cellularthiol and (and GSH) metabolism play a key role in enabling cells tore-establish homeostasis and retain reproductive integrity (resistance)in the presence of growth inhibiting drugs (MAPKi). Therefore, the goalwas to establish the role of reduced thiols in development of MAPKiresistance and simultaneously determine the clinical potential oftargeting thiol redox equilibrium in combination with MAPKi to improvethe efficacy of MAPKi to improve the efficacy of MAPKi in metastaticmelanoma patients.

The invention relates to the administration of a GSH synthesis inhibitorto a cancer patient along with one or more anti-cancer compositions toincrease the efficacy of the anti-cancer compositions by preventing drugresistance.

Definitions

As used herein, the term “GSH synthesis inhibitor” includes buthioninesulphoximine (BSO) and other compounds which inhibit the synthesis ofglutathione, including salts, isomers, analogs, and derivatives of suchcompounds.

As used herein, the term “buthionine sulphoximine” or “BSO” includessalts, isomers, analogs, and derivatives of BSO. BSO effectivelyinhibits y-glutamylcysteine synthetase by binding to the enzyme.

As used herein, the term “phenylbutyrate” includes salts, isomers,analogs and derivatives of phenylbutyrate. In certain embodiments,phenylbutyrate is Buphenyl® (sodium phenylbutyrate). Sodiumphenylbutyrate is used for chronic management of urea cycle disorders(UCDs). Its mechanism of action involves the quick metabolization ofsodium phenylbutyrate to phenylacetate. Phenylacetate then conjugateswith glutamine (via acetylation) to form phenylacetylglutamine, andphenylacetylglutamine is excreted by the kidneys. It has been observedthat sodium phenylbutyrate reduces Endoplasmic Reticulum (ER) stress.

The cellular response to ER stress is neither fully oncogenic norcompletely tumor suppressive. It involves complex signaling with manypathways. The relative importance of each pathway varies between cellsdepending on chronicity of ER stress, and on relative expression ofvarious associated proteins. As solid cancers grow, nutrients and oxygenrequired exceed capacity of existing vascular bed, which can triggerangiogenesis (development of new blood vessels) to get moreoxygen/nutrients to the cancers. Cancers, however, usually becomehypoxic and nutrient-depleted, and with the hypoxia leading to impairedgeneration of ATP. The low ATP levels compromise ER protein foldingwhich leads to ER stress. Thus, unfolded, and/or misfolded proteins areassociated with ER stress and cancer cells exist with higher levels ofER stress relative to health cells.

Potential outcomes as a consequence of ER stress include high rates ofprotein synthesis that would trigger increased expression of autophagy,which is cytoprotective during stress (liberates amino acids, andremoves damaged organelles). Another outcome would be an increasedtolerance to hypoxia, which would promote tumor growth. This would alsoincrease autophagy, promoting drug resistance. Thus, a successfultreatment would inhibit autophagy and promote cell death.

Sodium phenylbutyrate decreases ER Stress. Lowering ER stress preventstolerance to hypoxia and prevents cytoprotective autophagy (which leadsto drug resistance). Phenylbutyrate acts as a “chemical chaperone,”meaning it guides proper protein folding, and the presence of properlyfolded proteins lowers ER stress.

As used herein, the term “anti-cancer agent” includes therapeutic agentsthat kill cancer cells; slow tumor growth and cancer cell proliferation;and ameliorate or prevent one or more of the symptoms of cancer. Forexample, the term “anti-cancer agent” includes vemurafenib andtriphenylphosphonates (TPP). Vemurafenib (Zelboraf®) is a cancer growthblocker and is a treatment for advanced melanoma. Vemurafenib stops theproliferative effects of oncogenic BRAF protein. The standard method ofadministration is an oral tablet, administered 4× daily. Unfortunately,metastatic melanoma can resist vemurafenib treatment. Vemurafenib slowstumor progression for only about 5.3 months. As a result, finding aneffective treatment for metastatic melanoma is challenging.

For example, the term “anti-cancer agent” includes aTriphenylphosphonium (TPP) agent or derivative thereof that increasesreactive oxygen species (ROS) levels in cancer cell mitochondria, and apharmaceutically acceptable diluent or carrier. As used herein, the termtriphenylphosphonium (TPP) is any molecule containing atriphenylphosphine cation (+PPh₃) moiety. See, e.g., WO 2013/019975 andWO 2014/124384, which are incorporated by reference herein.

TPP salts can be reacted with alcohols, alkyl halides, and carboxylicacids, which allow them to be used as starting materials for thesynthesis of a large variety of chemical derivatives, e.g., XTPP agents.Charged molecules generally cannot pass through cell membranes withoutthe assistance of transporter proteins because of the large activationenergies need to remove of associated water molecules. In the TPPmolecules, however, the charge is distributed across the largelipophilic portion of the phosphonium ion, which significantly lowersthis energy requirement, and allows the TPP to pass through lipidmembranes. The phosphonium salts accumulate in mitochondria due to therelatively highly negative potential inside the mitochondrial matrix.The compositions of the present invention utilize XTPP agents that haveactivity in treating cancer cells, in that the XTPP agentspreferentially localize to cancer cells, as compared to the comparablenormal cells because cancer cells are often characterized by abnormalmitochondrial oxidative metabolism (Aykin-Burns N, Ahmad I M, Zhu Y,Oberley L W, and Spitz D R: Increased levels of superoxide and hydrogenperoxide mediate the differential susceptibility of cancer cells vs.normal cells to glucose deprivation. Biochem. J. 2009; 418:29-37. PMID:189376440) and altered mitochondrial membrane potential (Chen L B:Mitochondrial membrane potential in living cells, Ann. Rev. Cell Biol.1988; 4:155-81), relative to normal cells.

In certain embodiments, the TTP agent is 10-TTP or 12-TTP.

In certain embodiments, the anti-cancer agent is ipilimumab.

Compositions and Methods of Administration

The present invention provides a method for increasing the anticancereffects of a conventional cancer therapy (i.e., radio- and/orchemo-therapy) on cancerous cells in a mammal, comprising contacting thecancerous cell with an effective amount of a glutathione (GSH) synthesisinhibitor or a pharmaceutically acceptable salt thereof, andadministering an additional conventional cancer therapy modality. Incertain embodiments, the additional cancer therapy is chemotherapyand/or radiation. In certain embodiments, the GSH synthesis inhibitor ora pharmaceutically acceptable salt thereof and anti-cancer agent areadministered sequentially to a mammal rather than in a singlecomposition. In certain embodiments, the mammal is a human.

In certain embodiments of the methods described above, the compositiondoes not significantly inhibit viability of comparable non-cancerouscells.

In certain embodiments of the methods described above, the cancer isbreast cancer, prostate cancer, lung cancer, pancreas cancer, head andneck cancer, ovarian cancer, brain cancer, colon cancer, hepatic cancer,skin cancer, leukemia, melanoma, endometrial cancer, neuroendocrinetumors, carcinoids, neuroblastoma, glioma, tumors arising from theneural crest, lymphoma, myeloma, or other malignancies characterized byaberrant mitochondrial hydroperoxide metabolism. In certain embodiments,the cancer is the above cancers that are not curable or not responsiveto other therapies. In certain embodiments, the cancer is a melanoma. Incertain embodiments, the cancer is a glioma.

In certain embodiments of the methods described above, the tumor isreduced in volume by at least 10%. In certain embodiments, the tumor isreduced by any amount between 1-100%. In certain embodiments, the tumoruptake of molecular imaging agents, such as fluorine-18 deoxyglucose,fluorine-18 thymidine or other suitable molecular imaging agent isreduced by any amount between 1-100%. In certain embodiments, theimaging agent is fluorine-18 deoxyglucose, fluorine-18 thymidine orother suitable molecular imaging agent. In certain embodiments, themammal's symptoms (such as flushing, nausea, fever, or other maladiesassociated with cancerous disease) are alleviated.

Administration of a compound as a pharmaceutically acceptable acid orbase salt may be appropriate. Examples of pharmaceutically acceptablesalts are organic acid addition salts formed with acids that form aphysiological acceptable anion, for example, tosylate, methanesulfonate,acetate, citrate, malonate, tartrate, succinate, benzoate, ascorbate,α-ketoglutarate, and α-glycerophosphate. Suitable inorganic salts mayalso be formed, including hydrochloride, sulfate, nitrate, bicarbonate,and carbonate salts.

Pharmaceutically acceptable salts may be obtained using standardprocedures well known in the art, for example by reacting a sufficientlybasic compound such as an amine with a suitable acid affording aphysiologically acceptable anion. Alkali metal (for example, sodium,potassium or lithium) or alkaline earth metal (for example calcium)salts of carboxylic acids can also be made.

The GSH synthesis inhibitor and the anti-cancer agents can be formulatedas pharmaceutical compositions and administered to a mammalian host,such as a human patient in a variety of forms adapted to the chosenroute of administration, i.e., orally or parenterally, by intravenous,intramuscular, topical or subcutaneous routes.

Thus, the present compounds may be systemically administered, e.g.,orally, in combination with a pharmaceutically acceptable vehicle suchas an inert diluent or an assimilable edible carrier. They may beenclosed in hard- or soft-shell gelatin capsules, may be compressed intotablets, or may be incorporated directly with the food of the patient'sdiet. For oral therapeutic administration, the active compound may becombined with one or more excipients and used in the form of ingestibletablets, buccal tablets, troches, capsules, elixirs, suspensions,syrups, wafers, and the like. Such compositions and preparations shouldcontain at least 0.1% of active compound. The percentage of thecompositions and preparations may, of course, be varied and mayconveniently be between about 2 to about 60% of the weight of a givenunit dosage form. The amount of active compound in such therapeuticallyuseful compositions is such that an effective dosage level will beobtained.

In one embodiment of the invention, the GSH synthesis inhibitor is BSOand is administered intravenously in a dosage of between about 1.5-17g/m² as multiple infusion regimens.

The tablets, troches, pills, capsules, and the like may also contain thefollowing: binders such as gum tragacanthin, acacia, corn starch orgelatin; excipients such as dicalcium phosphate; a disintegrating agentsuch as corn starch, potato starch, alginic acid and the like; alubricant such as magnesium stearate; and a sweetening agent such assucrose, fructose, lactose or aspartame or a flavoring agent such aspeppermint, oil of wintergreen, or cherry flavoring may be added. Whenthe unit dosage form is a capsule, it may contain, in addition tomaterials of the above type, a liquid carrier, such as a vegetable oilor a polyethylene glycol. Various other materials may be present ascoatings or to otherwise modify the physical form of the solid unitdosage form. For instance, tablets, pills, or capsules may be coatedwith gelatin, wax, shellac or sugar and the like. A syrup or elixir maycontain the active compound, sucrose or fructose as a sweetening agent,methyl and propylparabens as preservatives, a dye and flavoring such ascherry or orange flavor. Of course, any material used in preparing anyunit dosage form should be pharmaceutically acceptable and substantiallynon-toxic in the amounts employed. In addition, the active compound maybe incorporated into sustained-release preparations and devices.

The active compound may also be administered intravenously orintraperitoneally by infusion or injection. Solutions of the activecompound or its salts can be prepared in water, optionally mixed with anontoxic surfactant. Dispersions can also be prepared in glycerol,liquid polyethylene glycols, triacetin, and mixtures thereof and inoils. Under ordinary conditions of storage and use, these preparationscontain a preservative to prevent the growth of microorganisms.

The pharmaceutical dosage forms suitable for injection or infusion caninclude sterile aqueous solutions or dispersions or sterile powderscomprising the active ingredient that are adapted for the extemporaneouspreparation of sterile injectable or infusible solutions or dispersions,optionally encapsulated in liposomes. In all cases, the ultimate dosageform should be sterile, fluid and stable under the conditions ofmanufacture and storage. The liquid carrier or vehicle can be a solventor liquid dispersion medium comprising, for example, water, ethanol, apolyol (for example, glycerol, propylene glycol, liquid polyethyleneglycols, and the like), vegetable oils, nontoxic glyceryl esters, andsuitable mixtures thereof. The proper fluidity can be maintained, forexample, by the formation of liposomes, by the maintenance of therequired particle size in the case of dispersions or by the use ofsurfactants. The prevention of the action of microorganisms can bebrought about by various antibacterial and antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, andthe like. In many cases, it will be preferable to include isotonicagents, for example, sugars, buffers or sodium chloride. Prolongedabsorption of the injectable compositions can be brought about by theuse in the compositions of agents delaying absorption, for example,aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activecompound in the required amount in the appropriate solvent with variousother ingredients enumerated above, as required, followed by filtersterilization. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and the freeze-drying techniques, which yield a powder ofthe active ingredient plus any additional desired ingredient present inthe previously sterile-filtered solutions.

For topical administration, the present compounds may be applied in pureform, i.e., when they are liquids. However, it may be desirable toadminister them to the skin as compositions or formulations, incombination with a dermatologically acceptable carrier, which may be asolid or a liquid.

Useful solid carriers include finely divided solids such as talc, clay,microcrystalline cellulose, silica, alumina and the like. Useful liquidcarriers include water, alcohols or glycols, or water-alcohol/glycolblends, in which the present compounds can be dissolved or dispersed ateffective levels, optionally with the aid of non-toxic surfactants.Adjuvants such as fragrances and additional antimicrobial agents can beadded to optimize the properties for a given use. The resultant liquidcompositions can be applied from absorbent pads, used to impregnatebandages and other dressings, or sprayed onto the affected area usingpump-type or aerosol sprayers.

Thickeners such as synthetic polymers, fatty acids, fatty acid salts andesters, fatty alcohols, modified celluloses or modified mineralmaterials can also be employed with liquid carriers to form spreadablepastes, gels, ointments, soaps, and the like, for application directlyto the skin of the user.

Examples of useful dermatological compositions that can be used todeliver the compounds of formula I to the skin are known to the art; forexample, see Jacquet et al. (U.S. Pat. No. 4,608,392), Geria (U.S. Pat.No. 4,992,478), Smith et al. (U.S. Pat. No. 4,559,157) and Wortzman(U.S. Pat. No. 4,820,508).

The dosage of the BSO or pharmaceutically acceptable salt thereof andthe anti-cancer agent will vary depending on age, weight, and conditionof the subject. Treatment may be initiated with small dosages containingless than optimal doses, and increased until a desired, or even anoptimal effect under the circumstances, is reached. In general, thedosage, involves escalating doses of BSO, from 5 to 17 gm/m², as amultiple infusion regimen.

Higher or lower doses, however, are also contemplated and are,therefore, within the confines of this invention. A medical practitionermay prescribe a small dose and observe the effect on the subject'ssymptoms. Thereafter, he/she may increase the dose if suitable. Ingeneral, the BSO or pharmaceutically acceptable salt thereof and theanti-cancer agent are administered at a concentration that will affordeffective results without causing any unduly harmful or deleterious sideeffects, and may be administered either as a single unit dose, or ifdesired in convenient subunits administered at suitable times.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. For example, thetherapeutic agent may be introduced directly into the cancer of interestvia direct injection. Additionally, examples of routes of administrationinclude oral, parenteral, e.g., intravenous, slow infusion, intradermal,subcutaneous, oral (e.g., ingestion or inhalation), transdermal(topical), transmucosal, and rectal administration. Such compositionstypically comprise the BSO or pharmaceutically acceptable salt thereofand the anti-cancer agent and a pharmaceutically acceptable carrier. Asused herein, “pharmaceutically acceptable carrier” is intended toinclude any and all solvents, dispersion media, coatings, antibacterialand anti-fungal agents, isotonic and absorption delaying agents, and thelike, compatible with pharmaceutical administration, and a dietaryfood-based form. The use of such media and agents for pharmaceuticallyactive substances is well known in the art and food as a vehicle foradministration is well known in the art.

Solutions or suspensions can include the following components: a sterilediluent such as water for injection, saline solution (e.g., phosphatebuffered saline (PBS)), fixed oils, a polyol (for example, glycerol,propylene glycol, and liquid polyethylene glycol, and the like),glycerine, or other synthetic solvents; antibacterial and antifungalagents such as parabens, chlorobutanol, phenol, ascorbic acid,thimerosal, and the like; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose. The properfluidity can be maintained, for example, by the use of a coating such aslecithin, by the maintenance of the required particle size in the caseof dispersion and by the use of surfactants. In many cases, it will bepreferable to include isotonic agents, for example, sugars, polyalcoholssuch as mannitol or sorbitol, and sodium chloride in the composition.Prolonged administration of the injectable compositions can be broughtabout by including an agent that delays absorption. Such agents include,for example, aluminum monostearate and gelatin. The parenteralpreparation can be enclosed in ampules, disposable syringes, or multipledose vials made of glass or plastic.

It may be advantageous to formulate compositions in dosage unit form forease of administration and uniformity of dosage. Dosage unit form asused herein refers to physically discrete units suited as unitarydosages for an individual to be treated; each unit containing apredetermined quantity of active compound calculated to produce thedesired therapeutic effect in association with the requiredpharmaceutical carrier. The dosage unit forms of the invention aredependent upon the amount of a compound necessary to produce the desiredeffect(s). The amount of a compound necessary can be formulated in asingle dose or can be formulated in multiple dosage units. Treatment mayrequire a one-time dose or may require repeated doses.

“Systemic delivery,” as used herein, refers to delivery of an agent orcomposition that leads to a broad biodistribution of an active agentwithin an organism. Some techniques of administration can lead to thesystemic delivery of certain agents, but not others. Systemic deliverymeans that a useful, preferably therapeutic, amount of an agent isexposed to most parts of the body. To obtain broad biodistributiongenerally requires a blood lifetime such that the agent is not rapidlydegraded or cleared (such as by first pass organs (liver, lung, etc.) orby rapid, nonspecific cell binding) before reaching a disease sitedistal to the site of administration. Systemic delivery of lipidparticles can be by any means known in the art including, for example,intravenous, subcutaneous, and intraperitoneal. In a preferredembodiment, systemic delivery of lipid particles is by intravenousdelivery.

“Local delivery,” as used herein, refers to delivery of an active agentdirectly to a target site within an organism. For example, an agent canbe locally delivered by direct injection into a disease site, othertarget site, or a target organ such as the liver, heart, pancreas,kidney, and the like.

The term “mammal” refers to any mammalian species such as a human,mouse, rat, dog, cat, hamster, guinea pig, rabbit, livestock, and thelike.

The terms “treat” and “treatment” refer to both therapeutic treatmentand prophylactic or preventative measures, wherein the object is toprevent or decrease an undesired physiological change or disorder, suchas the development or spread of cancer. For purposes of this invention,beneficial or desired clinical results include, but are not limited to,alleviation of symptoms, diminishment of extent of disease, stabilized(i.e., not worsening) state of disease, delay or slowing of diseaseprogression, amelioration or palliation of the disease state, andremission (whether partial or total), whether detectable orundetectable. “Treatment” can also mean prolonging survival as comparedto expected survival if not receiving treatment. Those in need oftreatment include those already with the condition or disorder as wellas those prone to have the condition or disorder or those in which thecondition or disorder is to be prevented.

In one embodiment of the invention, the GSH synthesis inhibitor isbuthionine sulfoximine (BSO). However, any GSH synthesis inhibitor isappropriate for use in the invention. In one embodiment of theinvention, the anti-cancer composition is a MAPK-pathway inhibitor(MAPKi) including, but not limited to, BRAF inhibitors (BRAFi), MEKinhibitors (MEKi), and ERK inhibitors (ERKi). Examples of BRAFi include,but are not limited to, vemurafenib, dabrafenib, and encorafenib.Examples of MEKi include, but are not limited to, refametinib,selumetinib, trametinib, and cobimetinib. Examples of ERKi include, butare not limited to, ERK1 and ERK2.

The GSH synthesis inhibitor may be administered concurrently with, priorto, or following administration of the one or more anti-cancer agents.In one embodiment, the GSH synthesis inhibitor may be administered up to10 days in advance of administration of the one or more anti-canceragents, or up to 10 days following the end of administration of the oneor more anti-cancer agents. GSH synthesis inhibitors are typicallyadministered intravenously (IV), but may also be administered by otherconventional means of administration where applicable, such as orally,subcutaneously (SQ), intramuscularly (IM), nasally, buccally, etc.

MAPK pathway inhibition has significantly improved progression-freesurvival (PFS) and overall survival (OS) of patients whose metastaticmelanoma malignancy is positive for specific mutations in the BRAFprotein. However, durable responses are rare, with many patientsexperiencing dramatic initial responses to treatment (responsive phase)that is followed by subsequent acquisition of drug resistance overseveral months (resistant phase). In vitro, MAPK pathway inhibition,initially reduces survival of BRAF mutant melanoma cells by almost 70%.However, almost 30% of the cell population exhibits a resistantphenotype (survives MAPK pathway inhibition). Furthermore, withcontinuous treatment, the resistant phenotype becomes dominant. BRAFinhibition is known to shift cellular metabolism from glycolysis tomitochondrial respiration (OXPHOS). To refine our understanding of thetiming of this metabolic shift, changes in metabolism and oxidativestate of live BRAF^(V600E) mutant melanoma cells (A375 and 451 Lu) wereexamined from the initiation of MAPKi treatments through the acquisitionof resistance (approximately 30 days). In order to quantify the shift inmetabolism, the ratio of basal metabolic oxygen consumption (BMOC) toextracellular acidification rate (ECAR, i.e., basal rate of glycolysis)by Seahorse SF6 extracellular flux analyzer was measured. Cells weretreated (continuously) with BRAFi (vemurafenib, 5 μM) alone and incombination with MEKi (cobimetinib, 0.1 μM) and measurements were madeusing live cells at pre-determined time points. Continuous treatment ofmelanoma cells A375 and 451Lu with MAPKi (BRAFi alone and in combinationwith MEKi) resulted in an increase in BMOC/ECAR as cells approached theresistant phase. The observed shift in cellular metabolism with theonset of resistance correlates with significant changes in themitochondrial and cellular oxidative state—suggesting a relationshipbetween the onset of resistance and oxidative state of the cell. Ascells became resistant to MAPKi, the fluorescence intensity of Mitosoxand DHE oxidation decreased to baseline levels indicatingre-establishment of new basal homeostasis and reproductive integrity.Considering the critical relationship between cellular oxidative stateand intracellular glutathione redox buffering, the changes in amount ofreduced GSH and the percent of GSH as GSSG were evaluated withcontinuous treatment of melanoma cells with MAPKi. We Continuoustreatment of melanoma cells with MAPKi resulted in a steady andsignificant increase in the concentration of reduced GSH and asignificant increase in the percent of total GSH as GSSG during theresponsive phase, but that these values decreased as the melanoma cellsacquired resistance to MAPKi.

To understand the role of thiol redox equilibrium in the development ofmelanoma MAPKi resistance involved pharmacologically disrupting thethiol (glutathione) redox balance in the presence of MAPKi. The effectof disrupting thiol redox equilibrium on development of MAPKi resistancewas quantified in terms of clonogenic survival of cells when treatedwith BRAFi (vemurafenib, 5 μM) alone and in combination withthiol-reducing agent 2-Mercaptoethanol (ME, 150 μM) or glutathionesynthesis inhibitor buthionine sulfoximine (BSO, 0.5 mM). Depletion ofGSH using BSO in the presence of MAPKi prevented the development ofresistance—i.e., the clonogenic survival of melanoma cells wasdramatically and significantly reduced when treated with the MAPKi incombination with buthionine sulphoximine (BSO). On the contrary,combining BRAFi with a biochemically-enhanced source of reduced thiols(i.e., 2-mercaptoethanol; ME) boosted the development of resistance(higher clonogenic survival) in melanoma cells A375 and 451Lu. Similareffects were observed when A375 cells were treated with ME incombination with BRAFi+MEKi. Interestingly, the effect of depletion ofreduced thiols (GSH) on acquisition of resistance was rescued byaddition of ME to the BSO/MAPKi combination, indicating that reducedcellular thiols play a key and vital role in the development of melanomaresistance to MAPKi.

2-Mercaptoethanol is a reducing agent that leads to reduction ofdisulphide bonds to produce thiols. Therefore, treating cells (A375)with combination of BRAFi and ME increased the cellular concentration ofGSH (reduced thiol), and treating cells with combination of BRAFi withBSO significantly decreased the cellular concentration of GSH to nearlyundetectable levels. The effect of eliminating GSH in vivo was tested bytreating mice (Athymic nu/nu) bearing A375 xenografts with rodent diet(AIN-76A) containing PLX4720 (vemurafenib, 416 mg/kg of diet) alone andin combination with BSO (15 mM in drinking water). No significantdifference in tumor growth rate was observed for untreated controls(FIG. 1 a ) and mice treated with BSO alone (FIG. 1 b ). As expected,significantly lower GSH concentrations were observed in tumor samplescollected from mice treated with BSO vs controls confirming thebioavailability of BSO in the tumor microenvironment. Tumor xenograftsin mice treated with the AIN-76 diet mixed with PLX4720 were responsiveinitially, but approximately 80% of these tumors acquired resistance toBRAFi treatment alone within 140 days (i.e., 20% complete responses;FIG. 2 ). On the contrary, the combination of BSO with PLX4720 producednearly 90% complete and durable tumor responses (FIG. 4 ). Body weightcomparison between untreated control mice and mice treated withcombination of BRAFi and BSO indicated that the combination was welltolerated. Tumor progression was defined as a 50% increase(approximately a two standard deviation increase) in tumor volume fromstudy initiation. Therefore, progression free survival (PFS) of micetreated with the combination of PLX4720 (vem, BRAFi) and BSO wassignificantly higher than mice treated with BRAFi alone (FIG. 3 ). Inaddition, the complete response rate and overall survival (OS) for thecohort treated with the combination was strikingly and significantlyhigher compared to untreated and PLX4720 treated groups (FIG. 4 ).

The following examples are offered to illustrate but not limit theinvention. Thus, it is presented with the understanding that variousformulation modifications as well as method of delivery modificationsmay be made and still are within the spirit of the invention.

Example 1

Methods

Cell Culture and Adenovirus Transfection

BRAF-mutant (BRAF^(V600E)) melanoma cell line A375 was purchased fromATCC. BRAF^(V600E) metastatic melanoma cell line 451Lu and itsvemurafenib-resistant derivative 451LuBR were obtained from the WistarSpecial Collection. A375 and 451Lu cells were cultured in high glucoseDMEM (Gibco) supplemented with 10% FBS (Gibco) and 1% Penstrep. 451LuBRcells were cultured in high glucose DMEM supplemented with 10% FBS, 1%Penstrep, and 5 μM vemurafenib (Selleckchem). A375-CG-LC3B cells wereobtained using a retroviral vector containing a coding sequence for amCherry-EGFP-LC3B tandem protein (pBABE-puro mCherry-EGFP-LC3B, #22418,Addgene). The retrovirus was packaged in GP2-293 cells byco-transfection of 1 μg of viral DNA constructs and 1 μg of envelopeplasmid (pVSV-G) in 20 μL PolyFect transfection reagent (Qiagen) for 48hours at 37° C., 5% CO₂. Virus-rich media was collected, aliquoted, andstored at −80° C. Cells were infected using the virus-rich media in DMEM(high glucose) for 12 h followed by incubation in complete media(high-glucose DMEM with 10% FBS) for 24 h. Infected cells were selectedusing 2 μg mL⁻¹ puromycin and transfection was confirmed usingmicroscopy and flow cytometry. A375-CG-LC3B cells were maintained inhigh glucose DMEM (Gibco) supplemented with 10% FBS (Gibco) and 1%Penstrep and 1 μg mL⁻¹ puromycin. All the cell lines were maintained in5% CO₂ at 37° C.

Drug Treatment

A375 and 451Lu cells were treated MAPK-pathway inhibitors (MAPKi),BRAF^(V600E) inhibitor (BRAFi) vemurafenib (Vem, 5 μM, Selleckchem)alone and in combination with MEK inhibitor (MEKi) cobimetinib (Cobi,0.1 μM, Selleckchem) for up to 35 days (4, 6, 10, 14, 21,2 8 and 35 daytime points). Buthionine sulfoximine (BSO, 0.5 mM, Sigma-Aldrich) wasused to inhibit glutathione (GSH) synthesis in the cells. Cells weretreated with β-mercaptoethanol (ME, 150 μM, Sigma-Aldrich). Further, toattenuate ER-stress, cells were treated with sodium 4-phenylbutyrate(PBA, 2 mM, Sigma-Aldrich). Cells were treated with Hydroxychloroquine(HCQ, 12 μM, Selleckchem) to inhibit autophagic flux. Dimethylsulfoximine (DMSO) and PBS were used as vehicle controls.

In Vitro Survival Assays

Clonogenic assays were conducted to quantify changes in the reproductiveintegrity of cells under experimental conditions described herein. Cellswere treated at 60-70% confluency with suitable vehicle controls andexperimental treatments. Total cell pool (floaters and adherent cells)were collected at pre-determined time points. Cells from the total cellpool were re-plated at single cell density in 60 mm dishes (Corning,tissue culture treated) in triplicates. The colonies were allowed toform over 10-14 days. Once visible colonies appeared, they were fixed,stained and counted to obtain the plating efficiency.

To obtain the percent live, non-apoptotic cells (survival percent),cells were plated in six-well dishes (80,000-100,000 cells well⁻¹). Theplates were treated in triplicate when the wells reached 60-80%confluency. The total cell pool was collected and centrifuged (1200 rpmfor 5 min). The cell pellets were washed twice with room temperatureDPBS. The washed cell pellet was re-suspended in Annexin V buffer(556454, BD Biosciences) and incubated with Annexin V Allophycocyanin(APC)-conjugated antibody, 1:250 (A35110, Thermo Fisher) and Hoechst33258 pentahydrate (4 μg mL⁻¹, Molecular Probes) for 10 min at roomtemperature. Samples were analyzed (50,000 events) using LSR II flowcytometer (Becton-Dickson). The flow readout was used to calculate thesurvival percent using Flow Jo software (V10.4).

Autophagic Flux

Autophagy was quantified as autophagic flux (A_(f)) using A375-CG-LC3Bcells using the formula:A _(f) =F _(mcherry) /F _(GFP)where F_(mcherry) is mcherry fluorescence measured using the 532 nmlaser and F_(GFP) is GFP fluorescence measured using the 488 nm laser onLSR II. 60-80% confluent six-well plates were treated in triplicateswith suitable vehicle controls or experimental treatments. Atpre-determined time points the total cell pool was collected,centrifuged (1200 rpm, 5 min), and washed (twice) with cold PBS. Thecell pellets were stained with apoptotic marker Annexin V and Hoechst33258 as described above. Samples were analyzed on an LSR II flowcytometer (BD). A_(f) was calculated for live, non-apoptotic (Hoechstnegative, Annexin V negative) population using the derived parameterfunction in Flow Jo software (V10.4).Transmission Electron Microscopy (TEM)

Transmission electron microscopy (TEM) is considered an accurate methodfor the detection and quantification of autophagy. The high resolutionof TEM was used to accurately quantify autophagy by quantifying the areacovered by autophagosomes (AFA). To quantify AFA, cells were culturedand appropriately treated in 60 mm dishes. 70-80% confluent dishes werefixed overnight with 2.5% glutaraldehyde in 0.1 M cacodylate bufferfollowed by rinsing in 0.1 M cacodylate buffer. Dishes were treated witha buffered 1% osmium tetroxide solution reduced with 1.5% potassiumferrocyanide for 30 min at room temperature post fixation followed by asecond wash with 0.1 M cacodylate buffer. The cells were then stained enbloc with 2.5% uranyl acetate. The staining was followed with a completedehydration using gradually increasing concentrations of ethanol to100%. Infiltration of Eponate 12 epoxy resin and ethanol was performedfor 1 hour at a 1:1 concentration followed by several changes of 100%resin. The dish was cured for 48 hours at 60 degree Celsius. A 5 mm×5 mmsquare grid was placed in the dish to transpose the lines onto the backof the cured dish. The squares on the grid were numbered beginning fromthe upper right-hand corner. The total number of squares on the dish wascounted and divided by four. Four random squares were chosen using arandom number generator chart. Selected squares were collected anden-face sections of 80 nm thickness were cut using a Leica UC-6ultramicrotome, that were collected on 75 mesh copper grids coated witha thin layer of formvar for stability. The grids were thencounterstained with 5% uranyl acetate for 2 minutes and Reynold's leadcitrate for 2 minutes. Samples were imaged using a JEOL 1230transmission electron microscope at 120 kV. Eight cells per section wereimaged based on pre-determined criteria and magnification. Stereology toobtain the AFA was accomplished using Area Fraction-Fractionator (thisinstrument was purchased with an NIH Shared Instrumentation Grant #1S10OD014165-01A1). A 7 μm×7 μm counting frame with a grid spacing of 0.5 μmwas used. The area covered by autophagosomes was differentiated from thetotal cell area by using different probes. The data was plotted as AFAin treated group normalized to untreated control groups.

Intra-Cellular Glutathione Levels

Cells were plated (300,000) in 100 mm dishes (Corning, Tissue culturetreated). 60-70% confluent dishes were treated with suitable vehiclecontrols or experimental treatments. The cells were washed with PBS andscraped in 100-200 μL of 5% 5-sulphosalicyclic acid (Sigma Aldrich). Thesamples were centrifuged at 5000 rpm for 5 min to precipitate protein.The supernatant was used to measure the total cellular glutathione (GSH)by applying the 5,5′-dithiobis-2-nitrobenzoic acid (DTNB) recyclingassay in which the rate at which the yellow color accumulates with theintroduction of DTNB is proportional to the amount of total glutathione(GSH (reduced)+GSSG (oxidized)) present measured by spectrophotometry(Beckman 800 spectrophotometer). Glutathione disulfide (GSSG) wasmeasured by adding 20 μl of a 1:1 mixture of 2-vinylpyridine and ethanolper 100 μl of sample and incubating for 2 h prior to assaying. Theprecipitated protein was re-suspended in 0.1 N NaOH, and protein levelsdetermined using the BCA Assay Kit (Thermo Scientific). Glutathionedeterminations were normalized to protein content of whole homogenates.

In-Vivo Studies: Athymic Nu/Nu Mouse Model

Female athymic nu/nu mice (age six weeks) were purchased from Envigo(previously Harlan Laboratories) and housed in the Animal Care Facilityat The University of Iowa (Iowa City, Iowa). All procedures wereapproved by The University of Iowa Institutional Animal Care and UseCommittee. All mice were naïve to treatments at the time of xenograft.For each xenograft, 1-3×10⁶ cells (451LuBR and A375) were implantedsubcutaneously in a 1:1 suspension of Matrigel (Corning) and PBS (flank)using a 27-gauge needle. For all in vivo studies, the mice were randomlyassigned to experimental groups post-implant. Animals developed tumorswithin 7-10 days post-implant. Initiation of treatments was standardizedto the time that tumor volume ([(longest length)×(smallestlength{circumflex over ( )}2)]/2) reached 80-100 mm³. The dose and modeof delivery for the drugs used is shown in Table 1:

TABLE 1 Mode of delivery for the drugs used. Drugs Mode of delivery BSODrinking Water PLX4720 AIN-76A rodent diet (vemurafenib, BRAFi)Vemurafenib PO (Orally) Cobimetinib PO Hydroxychloroquine (HCQ) PO4-phenylbutyrate (PBA) Intraperitoneal (IP)

The dose and mode of delivery for PBA was determined by analyzing thepharmacokinetics C-14 labeled PBA in mice (FIG. 5 ). These drugs wereadministered alone and in combination depending on the experimentalgroup. The mice were monitored for their state of health every day.Tumor volume and animal weights were measured twice per week. Mice wereeuthanized when the observed state of health reached the endpointaccording to the approved IACUC protocol or when the tumor volumereached 1500 mm³.

Statistics was conducted using the tumor sizes (mm³) that were obtainedperiodically throughout the experiments, resulting in repeatedmeasurements for each mouse. Linear mixed effects regression models wereused to estimate and compare treatment group-specific tumor growthcurves. Pairwise comparisons were performed to identify treatment groupdifferences in the growth curves. The Kaplan-Meier method was used toestimate the survival curves, and group comparisons were made using thelog rank test. Mice who developed ulcerations were censored at day ofeuthanasia. All tests were two-sided and carried out at the 5%significance level using SAS v9.4 (SAS Institute, Cary, N.C.).

Immunohistochemistry Staining for LC3B Protein Expression

Tumor tissue was collected from mice at day 60 (posthumously). Theparaffin embedded tumor tissue was cut into 5 μm sections. The sectionswere deparaffinized and rehydrated using the autostainer. Antigenretrieval was done in 10 mM citrate buffer (95° C., 650 watts). Membranepermeabilization was done using 0.1% Triton-X in PBS (10 min, roomtemperature). Hydrogen peroxide reductase was quenched by incubating theslides in 3% hydrogen peroxide (H₂O₂) (15 min at room temperature).Sections were incubated in 5% normal goat serum in PBS (pH 7.4) for 2 hat room temperature. Next, sections were incubated with primary LC3antibody (1:100, Rabbit-anti-LC3B (D11) XP antibody, Cell SignalingTechnology (3868)) at 4° C. overnight. Post primary antibody incubation,slides were washed with PBS (pH 7.4) (3×5 min). The tissue sections wereincubated with ImmPRESS anti-rabbit-H1RP conjugate secondary antibody(Vector Laboratory) (30 min at room temperature). Sections were washedas mentioned above; incubated with ImmPACT NovaRED kit (SK4805, Vectorlaboratories) chromogen. The chromogen solution was removed as soon asthe brown color was visible (˜2 min). Slides were washed in runningdistilled water for 10 min and counterstained with Harris hematoxylin(15 s). Slides were washed in distilled water (10 min). Lastly, theslides were dehydrated and mounted with Clear Permaslip and cover glass.The LC3B expression was imaged and quantified using a Leica Ariol SlideScanner.

Mitochondrial Oxygen Consumption & Extracellular Acidification Rate

Mitochondrial oxygen consumption rate (OCR) and ExtracellularAcidification Rate (rate of glycolysis; ECAR) were measured using aSeahorse XF96 flux analyzer system (Agilent). Cells were seeded in96-well Seahorse XF96 cell culture microplates (10,000-15,000 cellswell⁻¹) and treated at 60-70% confluency. At pre-determined time points,cells were washed, followed by 1 h incubation in pre-warmed SeahorseBioscience modified DMEM XF assay medium supplemented with 25 mM glucoseand 1 mM sodium pyruvate. OCR and ECAR were measured in real time bySeahorse Bioscience XF96 extracellular flux analyzer. Cell number inindividual wells was obtained using a standard hemocytometer. The rateswere normalized to a per cell basis for every time point (amol O₂ cell⁻¹s⁻¹).

Intra-Cellular Oxidative State by Flow Cytometry

The intra-cellular oxidative state was quantified using aredox-sensitive fluorescent probe, Dihydroethidium (DHE). In thepresence of reactive oxygen species (ROS), DHE is oxidized to formethidium cations; 2-hydroxyethidium or ethidium (depending on the typeof ROS), both of which can be detected at the excitation/emission maximaof 518/606 nm⁴⁰. Cells were plated in 6-well plates (80,000 cellswell⁻¹; 60-70% confluent wells; in triplicate). Cells were stained atspecific time points with DHE according to the manufacturer's protocol(D1168, Thermo Fisher Scientific). Briefly, treated cells wereharvested; washed with PBS containing 5 mM sodium pyruvate; andcentrifuged at 1200 rpm for 5 min. The cell pellets were resuspended andincubated in 500 μL of 10 μM DHE in PBS (containing 5 mM sodiumpyruvate) for 40 min at 37° C., in the dark. Subsequently, the sampleswere filtered through a 35 μm mesh filter into polystyrene 12×75 mm testtubes (352052, Falcon) and placed on ice. Hoechst 33342 was used as alive-dead discriminator. Cells treated with Antimycin A (10 μM) wereused as experimental positive controls. The samples were analyzed on anLSR II flow cytometer (Becton-Dickson). Fluorescence intensity wasrecorded for 10,000 Hoechst negative events and the Median FluorescenceIntensity (MFI) was quantified using FlowJo software (V10.4). Data fortreated groups was normalized to untreated control cells and plotted asnormalized median fluorescence intensity (NMFI).

All publications, patents and patent applications cited herein areincorporated herein by reference. While in the foregoing specificationthis invention has been described in relation to certain embodimentsthereof, and many details have been set forth for purposes ofillustration, it will be apparent to those skilled in the art that theinvention is susceptible to additional embodiments and that certain ofthe details described herein may be varied considerably withoutdeparting from the basic principles of the invention.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention are to be construed to cover boththe singular and the plural, unless otherwise indicated herein orclearly contradicted by context. The terms “comprising,” “having,”“including,” and “containing” are to be construed as open-ended terms(i.e., meaning “including, but not limited to”) unless otherwise noted.Recitation of ranges of values herein are merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range, unless otherwise indicated herein, and eachseparate value is incorporated into the specification as if it wereindividually recited herein. All methods described herein can beperformed in any suitable order unless otherwise indicated herein orotherwise clearly contradicted by context. The use of any and allexamples, or exemplary language (e.g., “such as”) provided herein, isintended merely to better illuminate the invention and does not pose alimitation on the scope of the invention unless otherwise claimed. Nolanguage in the specification should be construed as indicating anynon-claimed element as essential to the practice of the invention.

Embodiments of this invention are described herein. Variations of thoseembodiments may become apparent to those of ordinary skill in the artupon reading the foregoing description. The inventors expect skilledartisans to employ such variations as appropriate, and the inventorsintend for the invention to be practiced otherwise than as specificallydescribed herein. Accordingly, this invention includes all modificationsand equivalents of the subject matter recited in the claims appendedhereto as permitted by applicable law. Moreover, any combination of theabove-described elements in all possible variations thereof isencompassed by the invention unless otherwise indicated herein orotherwise clearly contradicted by context.

What is claimed is:
 1. A combination of a glutathione (GSH) synthesisinhibitor and an anti-cancer composition comprising vemurafenib, wherebythe GSH synthesis inhibitor is included in a dosage of from 1.5-17 g/m²buthionine sulphoximine (BSO).
 2. The combination of claim 1, whereinthe GSH synthesis inhibitor is administered simultaneously with theanti-cancer composition.
 3. The combination of claim 1, wherein the GSHsynthesis inhibitor and the anti-cancer composition are administeredsequentially.
 4. The combination of claim 1, wherein administration ofthe anti-cancer composition begins about 1 to about 10 days beforeadministration of the GSH synthesis inhibitor.
 5. The combination ofclaim 1, wherein administration of the GSH synthesis inhibitor beginsabout 1 to about 10 days before administration of the anti-cancercomposition.
 6. The combination of claim 1, wherein administration ofthe GSH synthesis inhibitor and administration of the anti-cancercomposition begin on the same day.
 7. The combination of claim 1,wherein the anti-cancer composition further comprises cobimetinib. 8.The combination of claim 1, wherein the anti-cancer compositioncomprises a derivative of triphenylphosphonium (TPP).
 9. The combinationof claim 1, wherein the anti-cancer composition comprises ipilimumab.10. A kit comprising a GSH synthesis inhibitor, a container, and apackage insert or label indicating the administration of the GSHsynthesis inhibitor with an anti-cancer composition for treatingmelanoma, whereby the GSH synthesis inhibitor is buthionine sulphoximine(BSO) in a dosage of from 1.5-17 g/m², and further providing that theanti-cancer composition comprises vemurafenib.
 11. A method for treatinga drug-resistant melanoma in a mammal, comprising administering to themammal that is resistant to MAPK pathway inhibitor and/or could acquireresistance to MAPK pathway inhibitors a combination of a GSH synthesisinhibitor and vemurafenib, whereby the GSH synthesis inhibitor and thevemurafenib are administered sequentially, and further providing thatthe GSH synthesis inhibitor comprises buthionine sulphoximine (BSO) in adosage of from 1.5-17 g/m².
 12. The method of claim 11 whereby the BSOis administered 1-10 days prior to the vemurafenib.
 13. The method ofclaim 11 whereby the vemurafenib is administered 1-10 days prior to theBSO.
 14. A method for treating a drug-resistant melanoma in a mammal,comprising administering to the mammal that is resistant to MAPK pathwayinhibitor and/or could acquire resistance to MAPK pathway inhibitors acombination of buthionine sulphoximine (BSO) and vemurafenib, andfurther providing that the BSO is administered in a dosage of from1.5-17 g/m2.